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1.  TargetingTumor MetabolismWith 2-Deoxyglucose in Patients With Castrate-Resistant Prostate Cancer and Advanced Malignancies 
The Prostate  2010;70(13):1388-1394.
A profound difference between cancer and normal tissues is the preferential utilization of glycolysis by cancer cells. To translate this paradigm in the clinic, we completed a phase I study of 2-deoxyglucose (2DG), and assessed 2DG uptake with fluorodeoxyglucose (FDG) positron emission tomography (PET) and the autophagy substrate p62 as a marker of 2DG resistance.
Patients received 2DG orally on days 1–14 of a 21-day cycle in cohorts of three in a dose-escalating manner. Correlative assessments included PET scans at baseline and day 2 and p62 protein in peripheral blood mononuclear cells as a potential marker of 2DG resistance.
The dose of 45 mg/kg was defined as the recommended phase II dose, secondary to dose-limiting toxicity of grade 3 asymptomatic QTc prolongation at a dose of 60 mg/kg. PK evaluation of 2DG revealed linear pharmacokinetics with Cmax 45 μg/ml (277 μM), 73.7 μg/ml (449 μM), and 122 μg/ml (744 μM) in dose levels 30, 45, and 60 mg/kg, respectively. Five of eight patients assessed with FDG-PET scanning demonstrated decreased FDG uptake by day 2 of therapy, suggesting competition of 2DG with FDG. Five of six patients assessed for p62 had a decrease in p62 at 24 hr.
These data support the safety of 2DG, defined 2DG PK, demonstrated the effect of 2DG on FDG-PET imaging, and demonstrated the feasibility of assessment of p62 as an autophagic resistance marker. These data support future studies of 2DG alone or in combination with approaches to abrogate autophagy.
PMCID: PMC4142700  PMID: 20687211
deoxyglucose; metabolism; prostate cancer; autophagy; p62
The Prostate  2012;72(12):10.1002/pros.22487.
Targeting multiple anti-apoptotic proteins is now possible with the small molecule BH3 domain mimetics such as ABT-737. Given recent studies demonstrating that autophagy is a resistance mechanism to multiple therapeutic agents in the setting of apoptotic inhibition, we hypothesized that hydroxychloroquine (HCQ), an anti-malarial drug that inhibits autophagy, will increase cytotoxicity of ABT-737.
Experimental Design
Cytotoxicity of ABT-737 and HCQ was assessed in vitro in PC-3 and LNCaP cells, and in vivo in a xenograft mouse model. The role of autophagy as a resistance mechanism was assessed by siRNA knockdown of the essential autophagy gene beclin1. ROS was measured by flow cytometry, and mitophagy assessed by the mCherry-Parkin reporter.
Induction of autophagy by ABT-737 was a mechanism of resistance in prostate cancer cell lines. Therapeutic inhibition of autophagy with HCQ increased cytotoxicity of ABT-737 both in vitro and in vivo. ABT-737 induced LC-3 and decreased p62 expression by immunoblot in cell lines and by immunohistochemistry in tumors in vivo. Assessment of ROS and mitochondria demonstrated that ROS production by ABT-737 and HCQ was a mechanism of cytotoxicity.
We demonstrated that autophagy inhibition with HCQ enhances ABT-737 cytotoxicity in vitro and in vivo, that LC-3 and p62 represent assessable markers in human tissue for future clinical trials, and that ROS induction is a mechanism of cytotoxicity. These results support a new paradigm of dual targeting of apoptosis and autophagy in future clinical studies.
PMCID: PMC3840901  PMID: 22241682
Prostate Cancer; Autophagy; Metabolism; Bcl-2; BH3; ABT-737; ABT-263
3.  Autophagy Suppresses RIP Kinase-Dependent Necrosis Enabling Survival to mTOR Inhibition 
PLoS ONE  2012;7(7):e41831.
mTOR inhibitors are used clinically to treat renal cancer but are not curative. Here we show that autophagy is a resistance mechanism of human renal cell carcinoma (RCC) cell lines to mTOR inhibitors. RCC cell lines have high basal autophagy that is required for survival to mTOR inhibition. In RCC4 cells, inhibition of mTOR with CCI-779 stimulates autophagy and eliminates RIP kinases (RIPKs) and this is blocked by autophagy inhibition, which induces RIPK- and ROS-dependent necroptosis in vitro and suppresses xenograft growth. Autophagy of mitochondria is required for cell survival since mTOR inhibition turns off Nrf2 antioxidant defense. Thus, coordinate mTOR and autophagy inhibition leads to an imbalance between ROS production and defense, causing necroptosis that may enhance cancer treatment efficacy.
PMCID: PMC3406086  PMID: 22848625
4.  Bcl-2 Modulation to Activate Apoptosis in Prostate Cancer 
Molecular cancer research : MCR  2009;7(9):1487-1496.
Apoptosis resistance is a hallmark of cancer linked to disease progression and treatment resistance, which has led to the development of anticancer therapeutics that restore apoptotic function. Antiapoptotic Bcl-2 is frequently overexpressed in refractory prostate cancer and increased following standard hormonal therapy and chemotherapy; however, the rationally designed Bcl-2 antagonist, ABT-737, has not shown single agent apoptosis-promoting activity against human prostate cancer cell lines. This is likely due to the coordinate expression of antiapoptotic, Bcl-2–related Mcl-1 that is not targeted by ABT-737. We developed a mouse model for prostate cancer in which apoptosis resistance and tumorigenesis were conferred by Bcl-2 expression. Combining ABT-737 with agents that target Mcl-1 sensitized prostate cancer cell lines with an apoptotic block to cell death in vitro. In mice in vivo, ABT-737 showed single agent efficacy in prostate tumor allografts in which tumor cells are under hypoxic stress. In human prostate cancer tissue, examined using a novel tumor explant system designated Tumor Tissue Assessment for Response to Chemotherapy, combination chemotherapy promoted efficient apoptosis. Thus, rational targeting of both the Bcl-2 and Mcl-1 mechanisms of apoptosis resistance may be therapeutically advantageous for advanced prostate cancer.
PMCID: PMC2855683  PMID: 19737977
5.  Autophagy Suppresses Tumorigenesis Through Elimination of p62 
Cell  2009;137(6):1062-1075.
Allelic loss of the essential autophagy gene beclin1 occurs in human cancers and renders mice tumor-prone suggesting that autophagy is a tumor-suppression mechanism. While tumor cells utilize autophagy to survive metabolic stress, autophagy also mitigates the resulting cellular damage that may limit tumorigenesis. In response to stress, autophagy-defective tumor cells preferentially accumulate p62/SQSTM1 (p62), endoplasmic reticulum (ER) chaperones, damaged mitochondria, reactive oxygen species (ROS), and genome damage. Moreover, suppressing ROS or p62 accumulation prevented damage resulting from autophagy defects indicating that failure to regulate p62 caused oxidative stress. Importantly, sustained p62 expression resulting from autophagy defects was sufficient to alter NF-κB regulation and gene expression and to promote tumorigenesis. Thus defective autophagy is a mechanism for p62 upregulation commonly observed in human tumors that contributes directly to tumorigenesis likely by perturbing the signal transduction adaptor function of p62 controlling pathways critical for oncogenesis.
PMCID: PMC2802318  PMID: 19524509
autophagy; beclin1; atg5; genomic instability; p62; NF- κB; DNA damage; cancer
6.  Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis 
Cancer cell  2006;10(1):51-64.
Defective apoptosis renders immortalized epithelial cells highly tumorigenic, but how this is impacted by other common tumor mutations is not known. In apoptosis-defective cells, inhibition of autophagy by AKT activation or by allelic disruption of beclin1 confers sensitivity to metabolic stress by inhibiting an autophagy-dependent survival pathway. While autophagy acts to buffer metabolic stress, the combined impairment of apoptosis and autophagy promotes necrotic cell death in vitro and in vivo. Thus, inhibiting autophagy under conditions of nutrient limitation can restore cell death to apoptosis-refractory tumors, but this necrosis is associated with inflammation and accelerated tumor growth. Thus, autophagy may function in tumor suppression by mitigating metabolic stress and, in concert with apoptosis, by preventing death by necrosis.
PMCID: PMC2857533  PMID: 16843265
7.  Glutamine deprivation stimulates mTOR-JNK-dependent chemokine secretion 
Nature Communications  2014;5:4900.
The non-essential amino acid, glutamine, exerts pleiotropic effects on cell metabolism, signalling and stress resistance. Here we demonstrate that short-term glutamine restriction triggers an endoplasmic reticulum (ER) stress response that leads to production of the pro-inflammatory chemokine, interleukin-8 (IL-8). Glutamine deprivation-induced ER stress triggers colocalization of autophagosomes, lysosomes and the Golgi into a subcellular structure whose integrity is essential for IL-8 secretion. The stimulatory effect of glutamine restriction on IL-8 production is attributable to depletion of tricarboxylic acid cycle intermediates. The protein kinase, mTOR, is also colocalized with the lysosomal membrane clusters induced by glutamine deprivation, and inhibition of mTORC1 activity abolishes both endomembrane reorganization and IL-8 secretion. Activated mTORC1 elicits IL8 gene expression via the activation of an IRE1-JNK signalling cascade. Treatment of cells with a glutaminase inhibitor phenocopies glutamine restriction, suggesting that these results will be relevant to the clinical development of glutamine metabolism inhibitors as anticancer agents.
Glutamine deprivation is currently being tested as a therapeutic strategy in cancer. Shanware et al. show that in cultured cells, glutamine deprivation stimulates IL-8 secretion by triggering endoplasmic reticulum stress, and suggest that the potential of this effect to influence tumour development should be examined.
PMCID: PMC4200525  PMID: 25254627

Results 1-7 (7)